1. Increased Internal and External Bacterial Load during Drosophila Aging without Life- Span Trade-Off 
Chunli Ren, Paul Webster, Steven E. Finkel, John Tower 
Cell Metabolism 
Volume 6, Issue 2, Pages (August 2007)
DOI: /j.cmet
Copyright © 2007 Elsevier Inc. Terms and Conditions

2. Figure 1 Drosophila Bacterial Load as a Function of Adult Age
(A and B) The total number of bacterial colony-forming units (cfu) cultured from the surface and the interior of the fly, plotted on log scale as mean ± SD.
(A) Total bacteria cultured aerobically.
(B) Total bacteria cultured anaerobically (same cohort as in [A]).
(C–G) Life-span analyses. Each panel presents an independent experiment. Oregon-R (O) and Canton-S (C) wild-type flies were cultured under control (Co) and axenic (Ax) conditions as indicated (C–E) or in the presence and absence of the indicated antimicrobials (F and G). Survival is plotted as a function of adult age in days. Each curve represents ≥125 flies. Median life span and SD was calculated for each group, and each experimental group was compared to the corresponding control by log-rank test. Results are presented to the right of each graph as the median, followed in parentheses by standard deviation, percent change, and p value. Antibiotics in (F): D = doxycycline, A = ampicillin, K = kanamycin; antifungals in (G): Nys = nystatin, Ket = ketoconazole. DMSO = vector for antifungals.
Cell Metabolism 2007 6, DOI: ( /j.cmet )
Copyright © 2007 Elsevier Inc. Terms and Conditions

3. Figure 2 Analysis of Fly Surface
Cells on the surface of old flies were detected using scanning electron microscopy (A–F) and bromophenol blue (BPB) staining of cells (G and H). Flies were cultured under either control conditions (A, B, C, E, and G) or axenic conditions (D, F, and H). The image in (B) is a higher magnification of the region of thorax bounded by the white box in (A). Note that BPB gives background staining of nervous (eye) tissue in both control (G) and axenic (H) flies. Scale bars: (A) = 200 μm, (B) = 20 μm, (C) and (D) = 10 μm, (E) = 5 μm, (F) = 10 μm.
Cell Metabolism 2007 6, DOI: ( /j.cmet )
Copyright © 2007 Elsevier Inc. Terms and Conditions

4. Figure 3
Expression of Antimicrobial Peptide Genes during Aging of Control and Axenic Flies
(A) Northern blot analysis of total RNA isolated from young (Y) and old (O) flies of Oregon-R and Canton-S strains, cultured under either control (Co) or axenic (Ax) conditions, as indicated. The northern blot was sequentially hybridized with probes specific for the indicated antimicrobial peptide (AMP) genes and the ribosomal protein 49 (Rp49) gene as a loading control.
(B) Quantification of northern blot data. Bars indicate mean ± SD.
(C) Semiquantitative real-time PCR analysis of AMP gene induction during aging in Oregon-R (Or-R) and Canton-S (CS) wild-type strains cultured under control and axenic conditions, as indicated. Values are normalized to Rp49 and plotted on log scale as mean ± SD. Methods are presented in Supplemental Data.
(D) Summary of results. Aging is known to decrease immune function and survival in Drosophila, humans, and other species (indicated by solid lines). In this study, microbial load and AMP gene expression were modulated by altering culture conditions and using antimicrobial drugs; this had little or no effect on survival (indicated by dashed lines). The data suggest that under the optimized conditions of the laboratory, life span is not limited by microbes; instead, decreased immune function leads to increased microbial load and increased AMP gene expression, and these effects occur in parallel with some other mechanism acting downstream of the aging mechanism (or mechanisms) and causing decreased survival.
Cell Metabolism 2007 6, DOI: ( /j.cmet )
Copyright © 2007 Elsevier Inc. Terms and Conditions